Method and agent for treating vulnerable plaque

ABSTRACT

A method and gene therapy agent for treating a vulnerable plaque associated with a blood vessel of a patient is disclosed. The method includes providing at least one gene therapy agent encoding at least one protein. The gene therapy agent is administered to a target cell population. The protein is expressed within the patient from a portion of the target cell population. The vulnerable plaque is modified as a result of the protein expression. The gene therapy agent includes at least one polynucleic acid encoding at least one protein. Administration of the gene therapy agent to a target cell population results in expression of the protein capable of modifying the vulnerable plaque.

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to the field of vasculartherapies. More particularly, the invention relates to a method andagent for treating a vulnerable plaque associated with a blood vessel ofa patient.

BACKGROUND OF THE INVENTION

Heart disease, specifically coronary artery disease (CAD), is a majorcause of death, disability, and healthcare expense. Until recently, mostheart disease was considered primarily the result of a progressiveincrease of hard plaque in the coronary arteries. This atheroscleroticdisease process of hard plaques leads to a critical narrowing (stenosis)of the affected coronary artery and produces anginal syndromes, knowncommonly as chest pain. The progression of the narrowing reduces bloodflow, triggering the formation of a blood clot. The clot may choke offthe flow of oxygen rich blood (ischemia) to heart muscles, causing aheart attack. Alternatively, the clot may break off and lodge in anotherorgan vessel such as the brain resulting in a thrombotic stroke.

Within the past decade, evidence has emerged expanding the paradigm ofatherosclerosis, coronary artery disease, and heart attacks. While thebuild up of hard plaque may produce angina and severe ischemia in thecoronary arteries, new clinical data now suggests that the rupture ofsometimes non-occlusive, vulnerable plaques causes the vast majority ofheart attacks. The rate is estimated as high as 60-80 percent. In manyinstances vulnerable plaques do not impinge on the vessel lumen, rather,much like an abscess they are ingrained under the arterial wall. Forthis reason, conventional angiography or fluoroscopy techniques areunlikely to detect the vulnerable plaque. Due to the difficultyassociated with their detection and because angina is not typicallyproduced, vulnerable plaques may be more dangerous than other plaquesthat cause pain.

The majority of vulnerable plaques include a lipid pool, necrotic smoothmuscle (endothelial) cells, and a dense infiltrate of macrophagescontained by a thin fibrous cap, some of which are only two micrometersthick or less. The lipid pool is believed to be formed because of apathological process involving low density lipoprotein (LDL),macrophages, and the inflammatory process. The macrophages oxidize theLDL producing foam cells. The macrophages, foam cells, and associatedendothelial cells release various substances, such as tumor necrosisfactor, tissue factor, and matrix proteinases. These substances canresult in generalized cell necrosis and apoptosis, pro-coagulation, andweakening of the fibrous cap. The inflammation process may weaken thefibrous cap to the extent that sufficient mechanical stress, such asthat produced by increased blood pressure, may result in rupture. Thelipid core and other contents of the vulnerable plaque (emboli) may thenspill into the blood stream thereby initiating a clotting cascade. Thecascade produces a blood clot (thrombosis) that potentially results in aheart attack and/or stroke. The process is exacerbated due to therelease of collagen and other plaque components (e.g., tissue factor),which enhance clotting upon their release.

The clear correlation between elevated serum cholesterol levels and thedevelopment of CAD is well established from numerous epidemiological andlongitudinal studies. Lipoproteins play a major role in plasmacholesterol transport and their levels are commonly measured todetermine risk for CAD. Unlike the other three major circulatinglipoproteins, the high density lipoprotein (HDL) is primarily involvedin the removal of cholesterol from peripheral tissues. HDL transportscholesterol back to the liver or to other lipoproteins by a processknown as reverse cholesterol transport. The “protective” role of HDL hasbeen confirmed in numerous in vitro and in vivo studies (Miller et al.,1977, Lancet 965-968; Whayne et al., 1981, Atherosclerosis 39:411-419;Badimon et al., 1992, Circulation 86:Suppl. III:86-94; and Koizumi etal., 1988, J. Lipid Res. 29:1405-1415). These studies have shown thatelevated levels of LDL are clearly associated with CAD (i.e., presumablythrough a role in vulnerable plaque formation) whereas high HDL levelsappear to confer cardiovascular protection.

Apolipoprotein A1 (Apo-A1) is the major component of the HDL particleand is thought to play an important role in HDL protection against CAD.Indeed, high plasma levels of Apo-A1 are associated with reduced risk ofCAD and presence of coronary lesions (Gordon et al., 1989, N. Eng. J.Med. 321:1311-1316; Gordon et al., 1989, Circulation 79:8-15; and Rubinet al., 1991, Nature 353:265-267). Apo-A1 has been sequenced andcomprises a single polypeptide chain of 243 amino acids (Brewer et al.,1978, Biochem. Biophys. Res. Commun. 80:623-630). The major functioningdomain of the Apo-A1 molecule is believed to be the 11 or 22 repeatingamino acids putatively forming an amphipathic helical formation (Segrestet al., 1974, FEBS Lett. 38:247-253). This structure presumable providesthe main biologic activities associated with Apo-A1 (e.g., lipid bindingand lecithin cholesterol acyl-transferase (LCAT) activation).

The apolipoprotein variant A1-Milano (Apo-A1 Milano) is the firstdescribed molecular variant of Apo-A1 (Franceschini et al., 1980, J.Clin. Invest. 66:892-900). It is characterized by a substitution ofarginine-173 with a cysteine residue (Weisgraber et al., 1983, J. Biol.Chem. 258:2508-2513). The mutant apolipoprotein is transmitted as anautosomal dominant trait and eight generations of carriers have beenidentified (Gualandri et al., 1984, Am. J. Hum. Genet. 37:1083-1097).The status of the Apo-AIM carrier individual is characterized by aremarkable reduction in HDL-cholesterol level. In spite of this, theaffected subjects do not apparently show any increased risk of arterialdisease; indeed, by examination of the genealogic tree it appears thatthese subjects are “protected” from atherosclerosis.

The mechanism of the possible protective effect of Apo AI-M in thecarriers seems to be linked to a modification in the structure of themutant apolipoprotein, with the loss of one alpha-helix and an increasedexposure of hydrophobic residues (Francheschini et al., 1985, J. Biol.Chem. 260:1632-1635). The loss of the tight structure of the multiplealpha-helices leads to an increased flexibility of the molecule, whichassociates more readily with lipids, compared to normal AI. Moreover,apolipoprotein/lipid complexes are more susceptible to denaturation,thus suggesting that lipid delivery is also improved in the case of themutant.

Strategies have been developed for treating patients at risk for CADwith lipid lowering drugs and Apo-A1 Milano preparations. The lipidlowering drugs (e.g., bile-acid binding resins, statins, niacin andnicotinic acid, fibrates, and oral estrogens) each has its own drawbacksand limitations in terms of efficacy, side-effects, and qualifyingpatient population. Although advances have been made in the large-scaleproduction of Apo-A1Milano for cardiovascular treatments, problemsremain in its therapeutic use. For example, systemic administration ofApo-A1Milano may require repeated treatments over a long time period toachieve desirable results. In addition, patients successfully treatedwith Apo-A1Milano may not benefit from the therapy for long periods oftime. As such, it would be desirable to provide an Apo-A1Milanotreatment strategy that overcomes the disadvantages associated with theprior art.

Gene therapy science has developed in recent years and provides apotential treatment for many disorders. In gene therapy, somatic cellsare modified in or ex vivo to express a gene corresponding to atherapeutically or diagnostically useful protein. The geneticinformation necessary to encode and express the protein is transferredinto the cells by a number of techniques including injection, directuptake, receptor-mediated uptake, electroporation, precipitation, andothers. In vivo gene therapy typically involves the direct transfer ofgenetic material into a target cell group within a patient's body.Injection, direct uptake, receptor-mediated uptake, intravenousadministration, and ingestion are generally used for this type oftherapy. Alternatively, ex vivo therapy may involve removing a group ofcells from the patient (e.g., “harvesting”) and transferring the geneticinformation in vitro followed by re-introduction of the modified cellsback into the patient. The ex vivo therapy may also involve transferringthe information to a variety of donor cells.

One area of gene therapy research relates to the circulatory system.Researchers have transferred genetic material to vascular tissues,including smooth muscle and endothelial cells. Engineered cells arecapable of secreting the transferred protein for a significant period oftime. For example, human adenosine deaminase was expressed in vivo byrat vascular smooth muscle cells for over six months (Lynch et al.,1993, Proc. Natl. Acad. Sci. USA 89:113842). Strategies are beingdeveloped to further prolong the stable expression of proteins by thetransduced cells. Although gene therapy holds promise for the treatmentof numerous vascular tissue disorders, effective therapies specific forvulnerable plaque lesions are still lacking. What is needed, therefore,is a vulnerable plaque treatment strategy that would take advantage ofthe benefits associated with gene therapy.

Accordingly, it would be desirable to provide a strategy for treatingvulnerable plaque that would overcome the aforementioned and otherdisadvantages.

SUMMARY OF THE INVENTION

One aspect according to the invention provides a method of treating avulnerable plaque associated with a blood vessel of a patient. Themethod includes providing at least one gene therapy agent encoding atleast one protein. The gene therapy agent is administered to a targetcell population. The protein is expressed within the patient from aportion of the target cell population. The vulnerable plaque is modifiedas a result of the protein expression.

The gene therapy agent may include a polynucleic acid such asdeoxyribonucleic acid and ribonucleic add. The gene therapy agent mayinclude a vector such as a plasmid, retrovirus vectors, adenovirusvectors, Herpes Simplex vectors, Semliki Forest Virus vectors, andSindbis virus vectors. The gene therapy agent may be administered byinjection, direct uptake, receptor-mediated uptake, intravenousadministration, ingestion, electroporation, and precipitation.

The gene therapy agent may be administered in vivo the patient. The invivo gene therapy may be administered with a balloon catheter device, bystenting the blood vessel adjacent the vulnerable plaque, and/or byinterstitial administration.

The gene therapy agent may be administered ex vivo the patient. The exvivo gene therapy may include harvesting the cell population from thepatient, selecting for the portion of target cells capable of expressingthe protein subsequent the administration of the gene therapy agent, andadministering the selected cells into the patient. The selected cellsmay be reintroduced into a pericardial space of the patient.Alternatively, the therapeutic protein may be introduced into allogeniccells using any acceptable technique, including plasmid delivery, any ofa variety of vector delivery techniques or any other suitable technique,where the transfected cells are thereafter introduced into the patientusing an immunoisolation device.

The protein may be a collagen isoform or an A1 apolipoprotein isoform,such as a mutant Milano isoform. The target cell population may bemuscle cells, vascular cells, hepatic cells, harvested patient cells,and/or donor cells. Expressing the protein may include secreting theprotein into a bloodstream, localized expression adjacent the vulnerableplaque, and/or modulating expression level with an expression cassette.Modifying the vulnerable plaque may include fibrous cap reinforcement,reduction of lipid pool size, modifying a lipid pool constitution,modifying an inflammation response, preventing vulnerable plaqueformation, and/or preventing vulnerable plaque enlargement.

Another aspect according to the invention provides a gene therapy agentfor treating a vulnerable plaque associated with a blood vessel of apatient. The gene therapy agent includes at least one polynucleic acidencoding at least one protein. Administration of the gene therapy agentto a target cell population results in expression of the protein capableof modifying the vulnerable plaque.

The polynucleic acid may be deoxyribonucleic acid and/or ribonucleicacid. The protein may be a collagen isoform or an A1 apolipoproteinisoform such as a mutant Milano isoform. The gene therapy agent mayfurther include a vector operably attached to the polynucleic acid. Thevector may be a plasmid, retrovirus vector, adenovirus vector, HerpesSimplex vector, Semliki Forest Virus vector, and Sindbis virus vector.The gene therapy agent may further include a liposome sheathing the genetherapy agent and/or an expression cassette encoded in the polynucleicacid.

The foregoing and other features and advantages of the invention willbecome further apparent from the following detailed description of thepresently preferred embodiments, read in conjunction with theaccompanying drawings. The detailed description and drawings are merelyillustrative of the invention, rather than limiting the scope of theinvention being defined by the appended claims and equivalents thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart of a method of treating a vulnerable plaqueassociated with a blood vessel of a patient, in accordance with oneembodiment of the present invention;

FIG. 2 shows a representative gene therapy vector according to oneembodiment of the present invention;

FIGS. 3A and 3B show the 729 base DNA and 243 amino acid sequences of arepresentative apolipoprotein-A1 isoform. The Milano isoform is producedby a C-to-T transition resulting in expression of a unique cysteineresidue (Cys173), the mutated locations shown by underline;

FIG. 4 is a schematic view of a balloon catheter used to administer agene therapy agent according to one embodiment of the present invention;and

FIG. 5 is a schematic view of a balloon catheter used to administer agene therapy agent according to another embodiment of the presentinvention.

DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

Referring to the drawings, wherein like reference numerals refer to likeelements, FIG. 1 is a flow chart of a method of treating a vulnerableplaque associated with a blood vessel of a patient, in accordance withone embodiment of the present invention. A vulnerable plaque isdistinguishable from other types of plaque, including hard plaques, bythe presence of a fibrous cap. The vulnerable plaque fibrous cap retainsa pool of lipids and other contents, which may be released into theblood vessel upon rupture. The released contents may form emboli thatcan lodge in a blood vessel thereby posing a risk to the patient.Vulnerable plaques, unlike hard plaques, are generally non-occlusive andas such, may not produce angina. The following description pertains totreatment of these vulnerable plaques.

Those skilled in the art will recognize that although the presentinvention is described primarily in the context of treating a vulnerableplaque while using specific gene therapy agents, the inventorscontemplate a broader method of application. Any number of treatmentagents capable of performing the prescribed function(s) may becompatible with the present invention. Furthermore, the treatment of thevulnerable plaque is not limited to the described methodology. Numerousmodifications, substitutions, and variations may be made to the methodand gene therapy agents while providing effective vulnerable plaquetreatment consistent with the present invention.

The term “target cell population,” as used herein, includes cells,tissues, and/or organs. The term “gene therapy agent”, as used herein,includes polynucleic add constructs which are single, double or triplexstranded, linear or circular, that are expressible or non-expressibleconstructs which can either encode for and express a functional protein,or fragment thereof, or interfere with the normal expression of a targetgene, gene transfer and/or expression vectors. The term “gene therapyagent”, may further include accessory molecules such as chemical drugs,protein drugs, nucleic acid drugs, combination chemical/protein/nucleicacid drugs, liposomes, and the like, which typically have a therapeuticeffect on vulnerable plaque and/or facilitate function of the genetherapy agent.

As shown in FIG. 1, vulnerable plaque treatment may begin by providing agene therapy agent (step 100). The composition of the gene therapy agentmay vary depending on the treatment strategy. In one embodiment, thegene therapy agent may include a polynucleic acid polymer of ribonucleicacid (RNA), which encodes one or more proteins (e.g., polypeptides).Specifically, the agent may include messenger RNA (mRNA) for directingtransitory protein expression. Use of mRNA typically provides proteinexpression for only about one day and does not require nuclearpenetration. As such, there is typically no genetic liability (e.g.,transformation, insertion, mutation, etc.).

In some situations, however, a more prolonged effect may be desiredwithout incorporation of the exogenous polynucleic acid into the hostcell genome. In order to provide such an effect, another embodiment ofthe invention provides a gene therapy agent including a polymer ofdeoxyribonucleic acid (DNA), which encodes one or more proteins.Non-replicating DNA sequences can be introduced into a target cellpopulation to provide production of a desired protein for periods ofabout up to six months and without integration of the DNA sequence intothe cell genome.

An even more prolonged effect may be achieved by introducing the DNAinto the target cell by means of a vector having the DNA sequenceinserted therein. Examples of vectors that may be adapted for use withthe present invention include, but are not limited to, a plasmid,retrovirus vectors, adenovirus vectors, Herpes Simplex vectors, SemlikiForest Virus vectors, and Sindbis virus vectors. Such vectors are wellknown to those skilled in the art and may be used to provide efficientintroduction of a polynucleic acid into the target cell.

The vector may include an expression cassette for modulating proteinexpression level. In the present description, the term “expressioncassette” refers to one or more polynucleotide sequences positionedwithin the vector for modulating the replication, transcription,translation, integration, secretion, and/or degradation of the vector,resulting mRNA, and/or resulting protein. In one embodiment, theexpression cassette may include a replicator sequence for enhancingreplication of the vector and ultimately increasing protein expressionlevel. Other examples of vectors with an expression cassette that may beadapted for use with the present invention include plasmid pBR322, withreplicator pMB1, or plasmid pMK16, with replicator ColE1.

In another embodiment, the expression cassette may include acell-specific promoter that permits protein expression only inpredetermined cells. In another embodiment, the vector may encode apolymerase for transcribing portions of the vector wherein thepolymerase may bind to specific recognition sites on the expressioncassette. The encoded polymerase may either recognize a generic promoterregion (i.e., common to many genes) or a unique promoter region (i.e.,specific to the vector).

In another embodiment, the expression cassette may include any number ofenhancer or repressors, which are known in the art for modulatingtranscription and, ultimately, protein expression level of a givenpolynucleotide sequence. In another embodiment, the expression cassettemay include encoded regions for binding of translation enhancer and/orrepressor proteins to any transcribed mRNA sequence. Protein translationlevel may be increased or decreased by the binding of the enhancer orrepressor proteins, respectively, to the mRNA. In another embodiment,the expression cassette may include sequence code that controlsintegration into the target cell genome, which may prolong proteinexpression. In another embodiment, the expression cassette may includesequence code that directs the secretion of the protein.

Those skilled in the art will recognize that a myriad of strategiesexist for modulating protein expression. Numerous such strategies may beadapted for use with the present invention. For example, proteinexpression level may be modulated by changing mRNA stability throughincorporation of AU-rich sequences in its 3′ untranslated region (UTR).Such sequences have been shown to accelerate mRNA degradation bystimulating the removal of the poly-A tail. As another example,recognition sites may be provided in the 3′ UTR for specificendonucleases to cleave the mRNA. Such degradation promoting sequencesmay be encoded in the polynucleic acid expression cassette of the genetherapy agent to modulate (e.g., decrease) protein expression.Alternatively, various strategies for increasing protein expression andenhancing mRNA stability are known in the art and may be adapted for usewith the present invention.

FIG. 2 shows a representative gene therapy vector 20 according to oneembodiment of the present invention. Vector 20, which in this case is adouble-stranded DNA plasmid, includes an expression cassette 22, encodedprotein 24, and two selection genes 26, 28. Selection genes may beprovided to aid gene therapy administration in terms of selecting thosecells that include vector and are capable of expressing the protein.Those skilled in the art will recognize that the configuration,constitution, and number of genes may vary greatly within a givenvector. The representative vector 20 is provided merely as one example.The design of the vector generally will vary depending on the method ofgene therapy administration.

In one embodiment, an immediate and long lived gene expression may beachieved by utilizing a gene therapy agent including a liposomalpreparation with both DNA and an RNA polymerase, such as the phagepolymerases T7, T3, and SP6. The liposome sheath or particle may alsoinclude an initial source of the appropriate RNA polymerase, by eitherincluding the actual enzyme itself, or alternatively, an mRNA coding forthat enzyme. When the liposome is introduced into the target cell, itdelivers the DNA and the initial source of RNA polymerase. The RNApolymerase, recognizing the promoters on the introduced DNA, transcribesboth genes, resulting in translation products comprising more RNApolymerase and the desired protein. Production of these materialscontinues until the introduced DNA, which may be in the form of aplasmid, is degraded. In this manner, production of the desired proteinin vivo may be achieved in a few hours and be extended for one month ormore.

The expression of the gene therapy agent encoded protein of the presentinvention typically has a therapeutic effect on vulnerable plaque. Inone embodiment, the protein may be a collagen isoform. Numerous isoformsof collagen are known in the art and may have beneficial effects on thetreatment of vulnerable plaque. For example, localized collagenexpression may reinforce or strengthen the fibrous cap thereby reducingchance of vulnerable plaque rupture.

In another embodiment, the encoded protein may be an A1 apolipoproteinisoform wherein the protein may also include the Apo-A1 Milano mutation.Exemplary DNA and protein sequences of human mature Apo-A1 are shown inFIGS. 3A and 3B, respectively. The mature protein consists of 243 aminoacids, as shown, whereby it may be formed by proteolitic cleavage of 24amino acids during its formation. The Milano isoform, or Apo-A1 M,includes a single base C-to-T transition thereby changing the codon ofCGC to TGC. The altered codon produces an amino acid change of Arg173 toCys173, putatively allowing dimerisation of the molecule.

In yet another embodiment, any number of proteins that may have abeneficial effect on vulnerable plaque treatment may be used with thepresent invention. It should be noted that that the specific proteinexamples described herein may vary (i.e., using an isoform of anotherspecies or of another predetermined design). In addition, the inventorscontemplate that numerous other protein may be adapted for use with thepresent invention and are not limited to the examples discussed herein.

After an appropriate gene therapy agent is provided, it is administeredto a target cell population (step 101). The method of administrationgenerally depends on whether the target cell population will be treatedin vivo or ex vivo. With in vivo gene therapy, the gene therapy agent isadministered to a patient using a variety of methods. The gene therapyagent transforms a target cell population within the patient followed byprotein expression. With ex vivo gene therapy, the gene therapy agent isadministered to a target cell population outside of the patient's body.The cells expressing the gene therapy protein(s) may then beadministered to the patient, e.g., through the use of one or more animmunoisolation devices. In vivo gene therapy generally requires fewersteps; however, ex vivo therapy can provide greater flexibility. Itshould be noted that, with either method, more than one gene therapyagent may be administered to provide vulnerable plaque treatment. Inaddition, each gene therapy agent may express more than one mRNA and/orprotein and, optionally, include one or more accessory drug or cofactor(e.g., therapeutic agent, enzyme, protein, lipid, nucleic acid, etc.).

With in vivo gene therapy, the agent may be administered to the patientby numerous methods including, but not limited to injection, directuptake, receptor-mediated uptake, intravenous administration, andingestion, all of which are known in the art. In one embodiment, thegene therapy agent may be injected (e.g., by needle or by sheer forcethrough the cell membrane) into target cell population of a giventissue. In another embodiment, the agent may be administered inproximity to the target cell population allowing eventual uptake throughdirect tissue contact (e.g., direct uptake). Gene therapy agentsassociated with liposomes and naked nucleic acids may be suited fordirect uptake administration. In another embodiment, the agent may beadministered by receptor-mediated uptake by the target cell population.Gene therapy agents associated with antibody coated liposomes orparticles may be suited for receptor-mediated uptake. Furthermore, theantibodies may be used to target the liposomes/particle to a specificcell target population, such as a certain tissue or organ. In anotherembodiment, the gene therapy agent may be administered intravenously tothe patient. In another embodiment, the gene therapy agent may beingested (e.g., orally or nasally) by the patient. Gene therapy agentsassociated with viral vectors may be suited for ingestion. Those skilledin the art will recognize that the gene therapy agent may beadministered to the patient by a variety of methods, including methodsnot here described.

The gene therapy agent may be delivered directly into the tissue massand/or into the interstitial space of tissues of the patient, includingthose of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus,heart, lymph, blood, blood vessel, bone, cartilage, pancreas, kidney,gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervoussystem, eye, gland, and connective tissue. The interstitial space of thetissues includes the periadventitial, intercellular, fluid,mucopolysaccharide matrix among the reticular fibers of organ tissues,elastic fibers in the walls of vessels or chambers, collagen fibers offibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. The agent may be preferably administered to andprotein expressed in persistent, non-dividing cells which aredifferentiated, although delivery and expression may be achieved innon-differentiated or less completely differentiated cells, such as, forexample, stem cells of blood or skin fibroblasts.

Muscle tissue provides an attractive gene therapy target as the cellsare typically competent in the ability to take up and expresspolynucleotides. This ability may be due to the singular tissuearchitecture of muscle, comprising multinucleated cells, sarcoplasmicreticulum, and transverse tubular system. Polynucleotides may enter themuscle through the transverse tubular system, which containsextracellular fluid and extends deep into the muscle cell. It is alsopossible that the polynucleotides enter damaged muscle cells which thenrecover. Muscle is also advantageously used as a site for the deliveryand expression of polynucleotides in a number of therapeuticapplications because humans have a proportionately large muscle masswhich is conveniently accessed by direct injection through the skin. Forthis reason, a comparatively large dose of a gene therapy agent can bedeposited in muscle by multiple injections, and repetitive injections,to extend therapy over long periods of time. In addition, the proceduremay be easily performed and may be carried out safely and withoutspecial skill or devices.

Vascular tissue may be advantageously chosen as the gene therapy targetas the expressed protein may have a direct therapeutic role on thevulnerable plaque. As such, the gene therapy protein may be expressedwithin the cells directly involved in and/or adjacent to the vulnerableplaque. In one embodiment, the gene therapy agent may be administered invivo with a balloon catheter device, which may provide localized andminimally invasive gene therapy administration. Such devices aretypically positioned within a blood vessel adjacent the vulnerableplaque prior to gene therapy agent delivery. The balloon catheter mayinclude features that facilitate the administration of the gene therapyagent. For example, the balloon catheter may include one or moredeployable needles for injection of the gene therapy agent into thevascular tissue. The catheter may include dual balloon portionspositioned upstream or downstream of the vulnerable plaque therebyallowing stoppage of blood flow that would interfere with intravasculargene therapy agent administration. Alternatively, the blood flow of thetarget vessel may be stopped by ligation. The catheter may include anexpandable stent deployed on the balloon. The stent may further includea drug coating that, for example, includes the gene therapy agent. Sucha drug coated stent would provide a prolonged and localizedadministration of the gene therapy and/or other therapeutic agents.

Numerous balloon catheter devices for administering therapeutic agentsand/or gene therapy agents are known in the art and may be adapted foruse with the present invention. By way of example, the device 30 shownin FIG. 4 and device 40 shown in FIG. 5 are balloon catheters that maybe used to deliver a gene therapy agent according to the presentinvention. In one embodiment, device 30 includes an elongated hollowflexible tubular member 31 attached to an operating head 32, which isshown inserted within a blood vessel 33 including a vulnerable plaque34. The operating head 32 contains a particle bombardment device 35disposed therein for discharging gene therapy agent-coated particlesthrough a discharge port (not shown).

The particles may be manufactured from a chemically inert substance suchas gold or diamond (either synthetic or natural) and may be coated withthe gene therapy agent of the present invention. The particles areaccelerated within the bombardment device 35 out of the discharge porttoward a predetermined target tissue, which in this case is the bloodvessel 33 wall. The particles carry enough momentum to penetrate thetarget tissue thereby allowing intracellular delivery of the genetherapy agent.

Device 30 may also include proximal and distal balloons 36, 37, whichare shown in an inflated state, girthing the tubular member 31 on eitherside of the operating head 32. A balloon inflation line (not shown)extends through the device 30 providing inflation pressures to theballoons 36, 37. Inflation of the balloons 36, 37 against the bloodvessel 33 walls serves to isolate a space 38 there between through whichthe gene therapy agent-coated particles may be discharged. The particlesmay exit through the operating head 32 and/or a side wall 39 of thedevice 30 toward the target cells. The device 30 may also includesuction and gas injection ports in communication with suction and gasinjection lines (not shown) thereby allowing any body fluids in thespace 38 isolated by the proximal and distal balloons 36, 37 to bedisplaced by gas. Displacement of fluid from the space 38 prior to genetherapy administration may better expose the targeted cells and minimizefrictional drag on the accelerated particles. Device 30 may be analogousto that described in U.S. Pat. No. 5,836,905 to Lemelson et al., whichis incorporated by reference herein.

In another embodiment, as shown in FIG. 5, device 40 includes a balloon41 disposed adjacent one end of an elongated hollow flexible tubularmember 42. Balloon 41, which is shown in an inflated state, defines aninterior chamber 43 in communication with a lumen 44 formed within thetubular member 42 to facilitate balloon 41 inflation and deflation. Aninner lumen 45 is sized to permit a guidewire to pass therethrough.Balloon 41 includes an outer peripheral surface 46, on which a pluralityof microencapsulated spheres 47 are impregnated in a coating material,which may be, but is not limited to, a hydrophilic material. Themicroencapsulated spheres 47, which are immersed in the coatingmaterial, include the gene therapy agent of the present invention. Themicroencapsulated spheres 47 may be extruded in the balloon 41 wallduring the manufacturing process. The microencapsulated spheres 47 maybe manufactured from a biologically inert material, such as a polymericmaterial, and are sized (e.g., on the order of 5 microns) and configuredto rupture upon application of a predetermined pressure caused byinflating the balloon 41. The microencapsulated spheres 47 arefabricated with a quantity of the gene therapy agent in accordance withknown techniques. The spheres 47 become embedded in a vessel wall 48when an initial pressure is communicated to the balloon 41, andthereafter rupture upon further inflation of the balloon 41 therebyadministering the gene therapy agent to the patient. Device 40 may beanalogous to that described in U.S. Pat. No. 6,129,705 to Grantz, whichis incorporated by reference herein.

Those skilled in the art will recognize that numerous other ballooncatheter devices may be adapted for use with the present invention,including a perforated balloon catheter, the pulse voltage needlecannula device described in U.S. Pat. No. 5,702,384 to Umeyama et al.,the balloon catheter with channel forming means described in U.S. Pat.No. 5,997,525 to March et al. as well as other injection-based catheterdevices, such as that shown in U.S. Pat. No. 5,112,305 to Barath or U.S.Pat. No. 5,354,279 to Hofling, each of which are incorporated both byreference herein. It should be noted that these balloon catheterdelivery devices may also be adapted to administer the gene therapyagent to tissues other than vascular tissue.

The in vivo gene therapy may include stenting the blood vessel adjacentthe vulnerable plaque. Numerous stent devices are known in the art andmay be adapted for use with the present invention. The stent typicallymaintains the inner diameter size of the vessel thereby reducing atendency of stentosis. Preferably, the stent includes a drug coating fordelivering the gene therapy agents and possibly other therapeuticagents. As such, a prolonged local administration of the gene therapyagent may be provided. The stent may be self-expanding orballoon-expandable and deployed by methods known in the art. In oneembodiment, the stent may include a polymer film drug coating analogousto that described in U.S. Pat. No. 5,700,286 to Tartaglia et al., whichis incorporated by reference herein. In another embodiment, the drugcoating may be prepared by a method analogous to that described in U.S.Pat. No. 6,358,556 to Ding et al., which is incorporated by referenceherein.

It should be noted that numerous devices known in the art, other thanballoon catheter devices and stents, may be adapted for in vivo genetherapy administration of the present invention. The minimally invasiveneedle catheter device described in U.S. Pat. No. 6,322,536 to Rosengartet al. is an example of one such device. It should also be noted thattissues other than those of muscle or blood vessel, and having a lessefficient expression of injected polynucleotides or a less proximaterelationship to the vulnerable plaque, may nonetheless be advantageouslyused as administration sites to produce therapeutic results. In thisapplication, and in many others, such as those in which an enzyme orhormone is the gene product, it is not necessary to achieve high levelsof protein expression in order to effect a valuable therapeutic result.Alternatively, it is possible that a certain tissue may be chosen on itsability to correctly process and secrete a “product” capable of having atherapeutic affect on vulnerable plaque. For example, the liver may beused because of its ability to secrete mature HDL particles, whichcontain the A1 apolipoprotein. Secreted HDL particles may enter thecirculation thereby providing a distal therapeutic effect on thevulnerable plaque.

With ex vivo gene therapy, the gene therapy agent is administered to atarget cell population outside of the patient's body. The cells are maybe harvested from the patient or obtained from a donor source. The genetherapy agent may be administered to the harvested and donor cells invitro. Alternatively, the gene therapy agent may be administered to thedonor cells in another organism. Those skilled in the art will recognizethat the nature and source of the harvested and donor cells may vary.For example, the donor cells may originate from another species and maybe engineered (i.e., to reduce immunoreactivity), of cloned or stem-lineorigin, and the like.

After the gene therapy agent is administered, the cells expressing thegene therapy protein (e.g., the “transduced” cells) may then beadministered to the patient. The gene therapy agent may be administeredto the target cell population by numerous methods including, but notlimited to, injection, direct uptake, receptor-mediated uptake,electroporation, and precipitation. In one embodiment, the gene therapyagent may be microinjected into a target cell, and the target cell maydivide to form a target cell population. In another embodiment, theagent may be administered to the cells by direct uptake (i.e., throughincubation of the gene therapy agent with the target cells). Genetherapy agents associated with liposomes (i.e., lipofection) and nakednucleic acids may be suited for direct uptake administration.

In another embodiment, the agent may be administered byreceptor-mediated uptake by the target cell population. Gene therapyagents associated with antibody coated liposomes or particles may besuited for receptor-mediated uptake. In another embodiment, the genetherapy agent may be administered by electroporation (i.e., subjectingthe target cell population to short bursts of electro shock). In anotherembodiment, the gene therapy may be administered by a precipitationprotocol known in the art. Those skilled in the art will recognize thatthe gene therapy agent may be administered to the target cell populationby a variety of methods, including methods not here described.

The administration of the gene therapy agent varies in efficiency, andis typically inefficient. As such, the protein may be expressed from asmall proportion of the target cell population. It is sometimesdesirable in the case of ex vivo administration to select for theportion of cells expressing the protein (i.e., by including one or moreselection markers) thereby increasing the “potency” of the administeredcells. The transduced cells may be administered to the patient by avariety of methods including, but not limited to, injection andimplantation. Numerous such methods are known in the art. In oneembodiment, transduced target cells may be injected with a needle orcannula device into a desired tissue or location of the patient. Morespecifically, the selected cells may be (re)introduced into apericardial space of the patient thereby providing localized genetherapy adjacent heart tissues. In another embodiment, a stent orvascular graft seeded with transduced endothelial cells may be implantedwithin a blood vessel adjacent the vulnerable plaque. Those skilled inthe art will recognize that the transduced cells may be administered tothe patient by a variety of methods, including methods not heredescribed.

After the gene therapy agent has been administered to the target cellpopulation and, optionally, the selected cells administered to thepatient, the protein is expressed within the patient from a portion ofthe target cell population (step 102). The expression level and durationof the protein may be influenced and controlled by a variety of factors.For example, as previously discussed, sequences provided in theexpression cassette may modulate replication, transcription,translation, integration, secretion, and/or degradation of the vector,resulting mRNA, and/or resulting protein. Furthermore, modulatorproteins encoded in a gene therapy agent may control expression leveland duration. Protein expression level and duration, however, need notbe modulated through an encoded sequence, but may be achieved byproviding other factors included with the gene therapy agent. Proteinregulators, polymerases, and other cofactors, for instance, may beincluded within a liposome. Protein expression may also be cell-specificby, for example, including a cell-specific promoter in the expressioncassette.

The gene therapy agent protein may be expressed in cells adjacent to orrelatively distant from the vulnerable plaque. In one embodiment, theprotein may be expressed is vascular tissue thereby providing alocalized expression adjacent the vulnerable plaque. This may beadvantageous for expression of, for example, a collagen protein therebyreinforcing the vulnerable plaque fibrous cap. In another embodiment,the protein may be expressed in a tissue distant from the vulnerableplaque. The protein(s) may then be secreted into the bloodstream therebyallowing a direct therapeutic effect on the vulnerable plaque. This maybe advantageous for expression of, for example, Apo-A1 Milano. Distanttissue expression may be used because certain protein(s) may only becapable of expression, packaging, and/or secretion in a certain tissue,such as the liver. The distant tissue may also provide more favorableexpression levels and/or feasibility of administration. For example,muscle tissue provides a large target mass as well as proximity to theskin to facilitate administration by injection.

The expressed protein may then have either a direct or indirect role inmodifying vulnerable plaque (step 103). Vulnerable plaque modificationincludes any changes of therapeutic benefit in the treatment ofvulnerable plaque. The changes include fibrous cap reinforcement (e.g.,thickening the fibrous cap by collagen expression), reduction of lipidpool size (e.g., enhancing cholesterol transport by Apo-A1 Milanoexpression), modifying a lipid pool constitution (e.g., by expressingproteins or factors such as anticoagulants secreted into the lipidpool), modifying an inflammation response (e.g., by expressinganti-inflammatory proteins), preventing vulnerable plaque formation(e.g., by Apo-A1 Milano expression), and preventing vulnerable plaqueenlargement (e.g., by Apo-A1 Milano expression). The gene therapy agentprotein may be expressed on either a short or long-term basis and as acurrent treatment of vulnerable plaque or as a prophylactic. In oneembodiment, Apo-A1 Milano, or another protein, may be expressed in apatient after the detection of vulnerable plaque as a current treatment.In another embodiment, Apo-A1 Milano, or another protein, may beexpressed in a patient as a preventative treatment, especially ffvulnerable plaque is suspected. In either case, protein expressionaccording to the present invention may be preferable to simpleadministration of the purified protein for vulnerable plaque treatment.Gene therapy offers the advantages of expression time and levelmodulation, tissue expressive targeting, and native protein packagingand secretion, all of which may not be provided by other methods.

While the embodiments of the invention disclosed herein are presentlyconsidered to be preferred, various changes and modifications may bemade without departing from the spirit and scope of the invention. Thegene therapy agents, devices and their method of use are not limited toany particular composition, design, type, or sequence. Moreover, theprocedure step order and method of achieving the same may vary withoutlimiting the utility of the invention. Upon reading the specificationand reviewing the drawings hereof, it will become immediately obvious tothose skilled in the art that myriad other embodiments of the presentinvention are possible, and that such embodiments are contemplated andfall within the scope of the presently claimed invention. The scope ofthe invention is indicated in the appended claims, and all changes thatcome within the meaning and range of equivalents are intended to beembraced therein.

1. A method of treating a vulnerable plaque associated with a bloodvessel of a patient, the method comprising: providing at least one genetherapy agent encoding at least one protein; administering the genetherapy agent to a target cell population; expressing the protein withinthe patient from a portion of the target cell population; and modifyingthe vulnerable plaque as a result of the protein expression.
 2. Themethod of claim 1 wherein the gene therapy agent comprises a polynucleicacid selected from a group consisting of deoxyribonucleic acid andribonucleic acid.
 3. The method of claim 1 wherein the gene therapyagent comprises a vector selected from a group consisting of a plasmid,retrovirus vectors, adenovirus vectors, Herpes Simplex vectors, SemlikiForest Virus vectors, and Sindbis virus vectors.
 4. The method of claim1 wherein the gene therapy agent administration comprises at least onetechnique selected from a group consisting of injection, direct uptake,receptor-mediated uptake, intravenous administration, ingestion,electroporation, and precipitation.
 5. The method of claim 1 wherein thegene therapy agent is administered in vivo the patient.
 6. The method ofclaim 5 wherein the in vivo gene therapy is administered with a ballooncatheter device.
 7. The method of claim 5 wherein the in vivo genetherapy comprises stenting the blood vessel adjacent the vulnerableplaque.
 8. The method of claim 5 wherein the in vivo gene therapy isadministered interstitially.
 9. The method of claims 1 wherein the genetherapy agent is administered ex vivo the patient.
 10. The method ofclaim 9 further comprising: harvesting the cell population from thepatient; selecting for the portion of target cells capable of expressingthe protein subsequent the administration of the gene therapy agent; andadministering the selected cells into the patient.
 11. The method ofclaim 10 wherein the selected cells are reintroduced into a pericardialspace of the patient.
 12. The method of claim 1 wherein the protein is acollagen isoform.
 13. The method of claim 1 wherein the protein is an A1apolipoprotein isoform.
 14. The method of claim 13 wherein the A1apolipoprotein is a mutant Milano isoform.
 15. The method of claim 1wherein the target cell population comprises cells selected from a groupconsisting of muscle cells, vascular cells, hepatic cells, harvestedpatient cells, and donor cells.
 16. The method of claim 1 whereinexpressing the protein comprises secreting the protein into abloodstream.
 17. The method of claim 1 wherein expressing the proteincomprises localized expression adjacent the vulnerable plaque.
 18. Themethod of claim 1 wherein expressing the protein comprises modulatingexpression level with an expression cassette.
 19. The method of claim 1wherein modifying the vulnerable plaque comprises a modificationselected from a group consisting of fibrous cap reinforcement, reductionof lipid pool size, modifying a lipid pool constitution, modifying aninflammation response, preventing vulnerable plaque formation, andpreventing vulnerable plaque enlargement.
 20. A gene therapy agent fortreating a vulnerable plaque associated with a blood vessel of apatient, the gene therapy agent comprising: at least one polynucleicacid encoding at least one protein wherein administration of the genetherapy agent to a target cell population results in expression of theprotein capable of modifying the vulnerable plaque.
 21. The gene therapyagent of claim 20 wherein the polynucleic acid selected from a groupconsisting of deoxyribonucleic acid and ribonucleic acid.
 22. The genetherapy agent of claim 20 wherein the protein is a collagen isoform. 23.The gene therapy agent of claim 20 wherein the protein is an A1 isoformof an apolipoprotein.
 24. The gene therapy agent of claim 23 wherein theA1 apolipoprotein is a mutant Milano isoform.
 25. The gene therapy agentof claim 20 further comprising a vector operable attached to thepolynucleic acid.
 26. The gene therapy agent of claim 25 wherein thevector is selected from a group consisting of a plasmid, retrovirusvectors, adenovirus vectors, Herpes Simplex vectors, Semliki ForestVirus vectors, and Sindbis virus vectors.
 27. The gene therapy agent ofclaim 20 further comprising a liposome sheathing the gene therapy agent.28. The gene therapy agent of claim 20 further comprising an expressioncassette encoded in the polynucleic acid.